Vitamin D3 deficient chick kidney microsomes in vitro metabolize 25-hydroxyvitamin D3 to yet structurally unidentified polar metabolites previously designated MIC-I and MIC-II3. Kidney microsomes of vitamin D3-repleted chicks could not be demonstrated to produce these metabolites when tritrium was the radioactive label in positions C-26 and C-27 of the substrate. However, when the 25-hydroxy-(26,27-14C)-vitamin D3 was the radioactive substrate, MIC-I or MIC-II production was independent of the vitamin D3 status of the chicks. These results suggest that under conditions of vitamin D3-sufficiency there is augmented sequential kidney metabolism of 25-hydroxyvitamin D3 to products with modified side-chains involving C-26 and (or) C-27. It is possible that this metabolism is responsible for the regulation of kidney cellular concentrations of 25-hydroxyvitamin D3. Another study concerns the iron-sulfur protein component of the kidney mitochondrial 25-OH-D3-1-hydroxylase. This protein has been purified to homogeneity by ion-exchange chromatography, gel-filtration, and preparative polyacrylamide electrophoresis. The yield is approximately 6.5 mg from a total of 15 g of starting mitochondrial protein. The protein has been characterized with respect to its Fe-S content, amino acid composition, molecular weight, sedimentation coefficient, and spectral properties. It is composed of a single polypeptide chain. Experiments for the production of antibodies in mice to the iron-sulfur protein are in progress.